Summary

The Rab family is part of the Ras superfamily of small GTPases. There are at least
60 Rab genes in the human genome, and a number of Rab GTPases are conserved from yeast
to humans. The different Rab GTPases are localized to the cytosolic face of specific
intracellular membranes, where they function as regulators of distinct steps in membrane
traffic pathways. In the GTP-bound form, the Rab GTPases recruit specific sets of
effector proteins onto membranes. Through their effectors, Rab GTPases regulate vesicle
formation, actin- and tubulin-dependent vesicle movement, and membrane fusion.

Protein family review

The compartmentalization of eukaryotic cells requires the transport of lipids and
proteins between distinct membrane-bounded organelles. This transport is tightly regulated
and typically occurs through transport vesicles that bud from a donor compartment
and fuse with an acceptor compartment. Rab GTPases ('Ras-related in brain' [1]), which belong to the Ras superfamily of small GTPases, have emerged as central
regulators of vesicle budding, motility and fusion. Like other regulatory GTPases,
the Rab proteins switch between two distinct conformations, one GTP-bound and the
other GDP-bound (see Figure 1). The GTP-bound conformation is generally regarded as 'active' [2], as this is the form that interacts with downstream effector proteins [3].

Figure 1. The Rab GTPase cycle. The Rab GTPase switches between GDP- and GTP-bound forms, which
have different conformations. Conversion from the GDP- to the GTP-bound form is caused
by nucleotide exchange, catalyzed by a GDP/GTP exchange factor (GEF). Conversion from
the GTP-to the GDP-bound form occurs by GTP hydrolysis, facilitated by a GTPase-activating
protein (GAP). The GTP-bound form interacts with effector molecules, whereas the GDP-bound
form interacts with Rab escort protein (REP) and GDP dissociation inhibitor (GDI).
Pi, inorganic phosphate.

Gene organization and evolutionary history

A recent analysis of the sequenced human genome and expressed sequence tags indicates
that humans have at least 60 different Rab family members (Figure 2) [4]. This must be regarded as a minimum estimate, as a small part of the genome still
remains to be sequenced and sequence annotations are still incomplete. Rab genes are
widely distributed over the human chromosomes [4]. Rab GTPases have been found in all eukaryotes investigated, including Saccharomyces cerevisiae (11 members), Caenorhabditis elegans (29 members) and Drosophila melanogaster (26 members) [4]; this large number and wide distribution underlines their importance in eukaryotic
cell biology. Most, but not all, of the yeast Rab GTPases (mostly called Ypt proteins)
have one or more putative mammalian homologs. In several cases, a mammalian Rab can
functionally replace its yeast counterpart, demonstrating conservation of functions
of the proteins within the eukaryotes.

Many Rab GTPases seem to be products of gene duplications, given that several subfamilies
of closely related Rab GTPases ('isoforms') with 75-95% sequence identity and overlapping
functions can be identified (Figure 2) [4]. Ten subfamilies - Rab1, Rab3, Rab4, Rab5, Rab6, Rab8, Rab11, Rab22, Rab27 and Rab40
- have also been defined based on distinct subfamily-specific sequence motifs, but
a number of Rab proteins cannot be grouped in these subfamilies [5,6] (see later). In general, Rab GTPases differ most in their carboxyl termini, which
have been implicated in subcellular targeting [7], whereas regions involved in guanine-nucleotide binding (see below) are most conserved.
Furthermore, mammalian Rab genes generally consist of several exons, and alternative
splicing has been reported [8].

Characteristic structural features

High-resolution structural information obtained by X-ray crystallography currently
available for four Rab GTPases: mouse Rab3a [9,10], Plasmodium falciparum Rab6 [11], and S. cerevisiae Ypt51p [12] and Sec4p [13]. The Rabs share a fold that is, in gross terms, common to all small GTPases of the
Ras superfamily. The fold consists of a six-stranded β sheet, comprising five parallel
strands and one antiparallel one, surrounded by five α helices. In these structures
the elements responsible for guanine nucleotide and Mg2+ binding, as well as GTP hydrolysis, are located in five loops that connect the α
helices and β strands. The amino-acid residues that come together in space to form
this active site are closely associated with either the phosphate groups of the bound
nucleotide and Mg2+ or the guanine base (Figure 3) and are highly conserved within the entire Ras superfamily; they can easily be used
to recognize any small GTPase.

Crystallographic analysis of p21Ras [14] and also recently of the yeast Rab Sec4p [13] in the GDP- and GTP-bound states shows that the proteins adopt two different conformations,
with the major nucleotide-induced differences occurring in regions denoted switch
I and switch II [15]. In the amino-acid sequence these switch regions are located in the loop 2 region
and the loop 4-α2-loop 5 region, respectively, and in the three-dimensional structure
they are found on the surface of the molecule. Numerous mutagenesis studies have shown
that the putative switch regions are crucial for the interaction of Rab proteins with
regulatory protein partners such as GDP/GTP exchange factors and GTPase-activating
proteins (see 'Localization and function'). Furthermore, the crystal structure of
a complex of Rab3a and its effector molecule rabphilin-3a shows that the switch regions
form an important part of the binding interface between the two proteins [10].

A recent extensive sequence-analysis study shows the presence of five distinct amino-acid
stretches that are characteristic of the Rab GTPases (Figure 3b) [6]. These so-called RabF regions (shown in red in Figure 3b) cluster in and around switch regions I and II and are suggested to provide a means
of unequivocally identifying Rab proteins. In addition, four regions (RabSF regions,
shown in dark blue in Figure 3b) have been identified that can be used to define the ten subfamilies of Rab GTPases
mentioned above [6,7]. The RabSF regions are on two different surfaces of the GTPases; they probably allow
specific binding of downstream effector molecules, which must recognize a specific
Rab or Rab subfamily in addition to detecting the nucleotide-binding state. In support
of this, the crystallographic study by Ostermeier and Brunger [10] showed that the well-characterized effector of Rab3a, rabphilin-3A, occupies two
major binding interfaces on the surface of the GTPase. These regions of Rab3a have
been named Rab complementarity-determining regions, and they involve both the switch
regions and Rab superfamily-specific motifs [6]. A model has therefore been suggested in which effectors and regulators bind both
to the RabF motifs in the switch I and II regions, to discriminate between active
and inactive conformations, and to RabSF regions for specificity.

Localization and function

Localization and regulation

Some Rabs are expressed ubiquitously in human tissues, whereas others are tissue-specific
(Table 1). Within cells, they are localized to the cytosolic face of distinct intracellular
membranes (see Figure 4 and Table 1). Their reversible membrane localization depends on the post-translational modification
of a cysteine motif at the very carboxyl terminus (CXXX, CC, CXC, CCXX or CCXXX where
X is any amino acid), with one or two highly hydrophobic geranylgeranyl groups [16]. This post-translational modification requires the initial recognition of the newly
synthesized Rab protein by a Rab escort protein (REP), which presents the Rab protein
to the geranylgeranyl transferase. REP then functions as a chaperone that keeps the
hydrophobic, geranylgeranylated Rab soluble and delivers it to the appropriate membrane
[17]. The specific targeting of Rab GTPases is thought to rely on membrane receptors
that recognize the complex between REP and specific Rabs [18], but so far no such receptors have been identified at the molecular level.

Figure 4. Intracellular vesicle transport pathways and localization of selected Rabs. The biosynthetic
pathway transports proteins from the endoplasmic reticulum (ER) through the Golgi
complex to the cell surface. In the trans-Golgi network (TGN), molecules can enter either constitutive secretory vesicles (CV)
or regulated secretory granules/vesicles (RV). In specialized cells melanosomes (M)
are a lysosome-related compartment that moves within the cell in an actin- and myosin-dependent
manner, generating pigmentation. Material internalized from outside the cell reaches
the early endosomes (EE) first and can be recycled back to the surface, either directly
or via a perinuclear recycling endosome (RE) compartment, or transported to late endosomes
(LE) and lysosomes. The biosynthetic and endocytic circuits (arrows) exchange material
at the level of the Golgi apparatus and the endosomal elements. The localization of
selected mammalian Rab proteins in the membrane compartments participating in these
transport processes is indicated.

The REP-associated Rab GTPases are thought to be in the GDP-bound form, whereas membrane
delivery is accompanied by the exchange of GDP with GTP, catalyzed by a GDP/GTP exhange
factor (GEF), and the release of REP [17]. Upon GTP hydrolysis, which is catalyzed by a GTPase-activating protein (GAP), the
Rab GTPase may be released from the membrane. This is mediated by Rab GDP-dissociation
inhibitor (GDI), which is capable of retrieving the geranylgeranylated, GDP-bound
Rab from intracellular membranes [19]. GDI has structural similarity to REP [20] and, like REP, GDI can present geranylgeranylated, GDP-bound Rab proteins to specific
membranes [21]. GDI, which is more abundant than REP, thus serves as a recycling factor that allows
several rounds of membrane association and retrieval of the Rab GTPases.

Function

A wealth of genetic and biochemical studies indicate that Rab GTPases function as
regulators of specific intracellular traffic pathways (for a recent review, see [3]). The key to their function is the recruitment of effector molecules that bind exclusively
to their GTP-bound form. Rab effectors are a very heterogeneous group of proteins:
some are coiled-coil proteins involved in membrane tethering or docking, while others
are enzymes or cytoskeleton-associated proteins. Two-hybrid screening for protein
interactions and affinity chromatography have revealed that the endosomal GTPase Rab5a
has several different effectors, and this is probably true for other Rabs as well
[22,23,24]. This means that a Rab GTPase may be capable of regulating several molecular events
at a restricted membrane location. For example, although initial studies showed that
Rab5a regulates endocytic vesicle tethering and fusion, more recent evidence suggests
that it also controls vesicle formation at the plasma membrane and microtubule-dependent
motility of endocytic structures [25,26,27]. Even though effectors for many Rab GTPases have been identified, the identification
and functional characterization of Rab effectors is still in an early phase. The introduction
of an efficient affinity-chromatography protocol promises to speed up the identification
of new effectors [24].

Important mutants

Gene knock-out studies in yeast have shown that some Rab GTPases are essential, whereas
others are dispensable [28]. The only mammalian Rab knockout so far, that of the neuronally expressed Rab3a,
resulted only in minor phenotypic changes in mice [29]. Several genetic diseases have been found to involve Rab GTPases or their interacting
proteins, however [30,31].

Griscelli syndrome is an autosomal recessive disorder that causes partial albinism.
There are two variants of this disease, one that is associated primarily with immunological
defects and one associated with neurological dysfunctions. The syndrome with immunological
defects is caused by missense mutations in the gene encoding Rab27a [32]. This GTPase regulates the movement of melanosomes to the cell periphery of melanocytes,
and it also regulates the secretion of lytic granules in cytotoxic T lymphocytes [33,34]. The lack of Rab27a thus causes pigment anomalies and dysfunctional T lymphocytes,
in agreement with the defects observed in the patients. The Griscelli syndrome with
neurological symptoms is caused by mutations in the gene encoding the motor protein
myosin Va [35], a putative Rab27a effector that drives the peripheral distribution of melanosomes
along actin filaments [33]. As myosin Va does not participate in the exocytosis of lytic granules, the inactivation
of this protein does not lead to immunological symptoms.

Choroideremia is an X-linked disease that involves the degeneration of the retinal
pigment epithelium and the adjacent choroid and retinal photoreceptor cell layers,
leading to blindness. The gene mutated in choroideremia is one of the two REP isoforms,
REP-1 [36]. Although the other isoform, REP-2, seems to be sufficient for the geranylgeranylation
of all Rab GTPases in all tissues except for the retinal pigment epithelium, REP-1
is essential for the efficient geranylgeranylation of Rab27a in this tissue. Thus,
a lack of REP-1 leads to a lack of functional Rab27a specifically in the retinal pigment
epithelium [37]. The degeneration of this epithelium and its adjacent layers may be due to deficient
melanosome transport and consequently a lack of protection against harmful light exposure.

A subgroup of patients with X-linked nonspecific mental retardation have mutations
in the gene for one of the GDI isoforms, GDI-α [38]. This isoform is particularly abundant in the brain, and dysfunctional membrane
recycling of one or more Rab GTPases in brain synapses, leading to aberrant neurotransmission,
is likely to underly the symptoms in this disease.

Frontiers

The Rab GTPases are a large family of proteins with a variety of regulatory functions
in membrane traffic. The central role of these proteins has become clear during the
past decade, as part of the progress in understanding in detail the mechanistic principles
of transport vesicle formation, movement, and fusion. Sequencing of the human genome
has allowed us to realize the diversity of the Rab gene family, though the functions
of a majority of the gene products are unknown. The availability of complete genomic
sequences, as well as important advances in molecular and cell biological methods,
promise to bring a significant progress in our understanding of Rab function in the
near future.

At the molecular level, the identification of novel GAPs, GEFs and effectors will
yield information about the regulation of Rab GTPases and the molecular complexes
they control. Crosstalk with regulatory mechanisms involving other members of the
Ras GTPase superfamily is already becoming apparent. A key question concerns the targeting
of the Rab GTPases. Which 'receptor' molecules determine their specific intracellular
distributions? A combination of biochemical and genetic approaches will hopefully
illuminate this issue.

At the level of the membrane, several aspects of Rab GTPase function remain to be
clarified. Are Rab GTPases confined to restricted membrane domains [3] and, if so, how is this determined? Furthermore, how do Rab GTPases and their effectors
regulate membrane budding, motility and fusion? With respect to membrane fusion, the
role of Rab effectors as membrane tethers is already being revealed, and it seems
realistic to expect that Rab-dependent membrane fusion may be reconstituted in vitro from purified components in the near future.

Finally, comprehending the ways in which the regulatory actions of Rabs intertwine
with cell-signaling cascades and developmental processes is an enormous task for cell
biologists. Here, the natural mutant models provided by human genetic diseases that
have defects in Rabs or their auxiliary proteins, as well as the novel genome-wide
approaches for gene expression analysis, will be instrumental.

Acknowledgements

We are grateful to Tapani Ihalainen for help in preparing Figure 4. This work was supported by the Research Council of Norway (H.S.), the Norwegian
Cancer Society (H.S.), the Novo-Nordisk Foundation (H.S.), the Academy of Finland
(grants 45817, 49987 and 50641 to V.M.O.), and the Sigrid Juselius Foundation (V.M.O.).

References

Touchot N, Chardin P, Tavitian A: Four additional members of the ras gene superfamily isolated by an oligonucleotide
strategy: molecular cloning of YPT-related cDNAs from a rat brain library.

Proc Natl Acad Sci USA 1987, 84:8210-8214.

Describes the identification of the first mammalian Rab GTPases, and the term 'Rab'
is introduced.

Using information from expressed sequence tags and the recently published genome sequences,
this paper describes a bioinformatic analysis of several protein families involved
in membrane traffic, including Rab GTPases.

Pereira-Leal JB, Seabra MC: The mammalian Rab family of small GTPases: definition of family and subfamily sequence
motifs suggests a mechanism for functional specificity in the Ras superfamily.

J Mol Biol 2000, 301:1077-1087.

This outstanding report contains extensive sequence comparisons within the Rab family
and analysis of the results along with the information available from crystallography
studies. The authors define sequence motifs that can be used to identify proteins
belonging to the Rab family, and further, to specify subfamilies. A model is presented
in which an effector, upon binding to a Rab, recognizes both Rab family-specific (switch)
motifs to discriminate between the nucleotide-bound states, and simultaneously subfamily-specific
regions that confer specificity on the interaction.

Describes the alternative splicing of the Rab6a transcript and the resulting expression
of two forms (Rab6a and Rab6a') that differ by three amino acids flanking one of the
GTP-binding regions. The two splice forms have different abilities to bind effectors
and to regulate intra-Golgi traffic.

Reports the first high-resolution structure of a Rab GTPase, that of Rab3a complexed
with the GTP analog GppNHp. The analysis reveals structural determinants that stabilize
the active conformation and regulate the GTPase activity of Rab proteins.

This important study reports the structure of Rab3a bound to the effector domain of
rabphilin-3A, an interacting partner suggested to mediate the regulatory action of
the Rab on downstream targets. The work sheds light on the specific determinants of
interaction between a Rab in the active conformation and its effectors.

Reports the crystal structure of P. falciparum Rab6 and its comparison with Rab3a. The work reveals a high degree of conservation
of the core fold and also suggests that the switch mechanism is highly similar between
the two proteins.

A report of the first crystal structure of a yeast Rab GTPase, that of Ypt51p, a regulator
of endocytic events, including comparisons with other closely related Rab proteins
to pinpoint determinants for specific effector binding and for fine tuning the intrinsic
rate of GTP hydrolysis.

Stroupe C, Brunger AT: Crystal structures of a Rab protein in its inactive and active conformations.

J Mol Biol 2000, 304:585-598.

This important study reports the first crystal structures of a Rab GTPase, the S. cerevisiae Sec4p, in both GDP- and GTP-bound conformations. The analysis allows precise identification
of the switch regions and provides detailed information on the mechanisms regulating
the Rab GTPase cycle.

Describes the characterization and partial purification of a membrane protein that
acts as a GDI displacement factor for Rab5, Rab7 and Rab9. This factor is likely to
participate in the specific targeting of these Rab GTPases to endosomes.

The crystal structure of GDI-α at 1.8Å resolution. The structure consists of a large
multi-sheet domain I and a smaller α-helical domain II. Sequence-conserved regions
between GDI and REP are clustered on one side of the structure.

A purified Rab5-GDI complex was used to demonstrate that GDI can deliver Rab GTPases
to specific membranes and that membrane association of the Rab GTPase is accompanied
by GDI release and nucleotide exchange.

Rabaptin-5, a cytosolic coiled-coil protein, was found to interact specifically with
Rab5-GTP and to be recruited to endosomes containing Rab5-GTP. Rabaptin-5 is required
for Rab5-dependent endosome fusion.

EEA1 was identified as a Rab5 effector on early endosomes. Its recruitment to endosome
membranes and function in endocytic membrane fusion require binding to Rab5-GTP as
well as phosphatidylinositol 3-phosphate.

An affinity column with immobilized Rab5-GTP was used to isolate Rab5 effectors from
brain cytosol. As many as 22 different proteins are specifically retained on this
column, indicating that Rab5 has many effectors. One of these effectors, EEA1, could
completely substitute for cytosol in an in vitro endosome fusion assay.

Rab5-GDI was identified as a complex required for the formation of plasma membrane-derived
clathrin-coated vesicles in semi-intact cells. This is the first evidence that Rab5
plays a direct role in vesicle formation.

Video microscopy demonstrated that GFP-Rab5-positive structures move along microtubules
in vivo. In vitro, Rab5 was found to stimulate the association of endosomes with microtubules and the
minus-end-directed transport of endosomes. Rab5-dependent endosome motility depends
on the phosphatidylinositol 3-kinase hVPS34.

The first (and so far only) knockout of a mammalian Rab GTPase in mice. Rab3a-/- mice are viable and show no striking phenotype, but electrophysiology reveals minor
differences in Ca2+-induced synaptic vesicle exocytosis compared to Rab3a+/+ mice.

Provides evidence for the role of Rab27a in the peripheral distribution of melanosomes
in melanocytes, and for myosin Va as a Rab27a effector. Rab27a was found to colocalize
with myosin Va on melanosomes in melanoma cells. Dominant-negative Rab27a mutants
have a peri-nuclear distribution of melanosomes. Rab27a and myosin Va were found to
coimmunoprecipitate.

Shows the importance of Rab27a for the function of cytotoxic T lymphocytes. Lymphocytes
from ashen mice with a loss-of-function mutation in the Rab27a gene show reduced polarization
and reduced release of cytotoxic granules upon stimulation.

Identifies Rab27 as a Rab GTPase that is inefficiently geranylgeranylated in choroideremia,
in the absence of REP-1. Rab27 is found in the retinal pigment epithelium, which is
affected in choroideremia.